observing single molecules complexing with cucurbit[7]uril … · abstract: in recent years,...

Observing Single Molecules Complexing with Cucurbit[7]urilthrough Nanogap Surface-Enhanced Raman SpectroscopyDaniel O. Sigle,†,¶ Setu Kasera,‡,¶ Lars O. Herrmann,† Aniello Palma,‡ Bart de Nijs,† Felix Benz,†

Sumeet Mahajan,† Jeremy J. Baumberg,*,† and Oren A. Scherman*,‡

†Nanophotonics Centre, Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.‡Melville Laboratory for Polymer Synthesis, Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, U.K.

*S Supporting Information

ABSTRACT: In recent years, single-molecule sensitivityachievable by surface-enhanced Raman spectroscopy (SERS)has been widely reported. We use this to investigatesupramolecular host−guest chemistry with the macrocyclichost cucurbit[7]uril, on a few-to-single-molecule level. Ananogap geometry, comprising individual gold nanoparticleson a planar gold surface spaced by a single layer of molecules,gives intense SERS signals. Plasmonic coupling between theparticle and the surface leads to strongly enhanced opticalfields in the gap between them, with single-molecule sensitivityestablished using a modification of the well-known bianalytemethod. Changes in the Raman modes of the host moleculeare observed when single guests included inside its cavityinternally stretch it. Anisotropic intermolecular interactions with the guest are found which show additional distinct features inthe Raman modes of the host molecule.

Single-molecule spectroscopy techniques have made re-markable contributions in understanding molecular chem-istry, revealing details about molecule−surface interactions,molecular geometries, and dynamic processes.1,2 Surface-enhanced Raman spectroscopy (SERS) with its specificmolecular fingerprinting capability is a powerful analyticaltool, requiring only simple experimental setups and samplehandling. By placing molecules in strong plasmonic fields, theinherently weak Raman scattering cross sections can bedramatically enhanced. This enables detecting substances atthe single-molecule level.3−6 Until now, single-molecule SERShas been primarily limited to detection and distinction betweendifferent chemical species. With the advent of analytical tools atsingle-molecule sensitivity,2 characterization of intermolecularinteractions has also drawn interest.7−9 It is therefore importantto extend the capabilities of SERS to investigate single-moleculechemical interactions.A powerful plasmonic construct with a strong field

confinement can be realized with the “nanoparticle on mirrorgeometry” (NPoM),10−12 where a gold nanoparticle (AuNP) isspaced above a flat Au surface by a single layer of molecules,establishing a plasmonic gap of only a few nanometers. Becausethe AuNP induces image charges within the metallic surface,plasmonic coupling similar to a nanoparticle dimer is created(Figure 1a,b). This self-assembled system thus creates a robustand reproducible plasmonic sensing platform for SERS withextremely high field localization and is several orders ofmagnitude below the classical diffraction limit. Due to the

strong confinement of the generated field enhancement, theinterrogated volume extends13 over only a few cubic nanome-ters and has shown promise in enabling down to single-molecule resolution.14,15

We choose the macrocyclic host molecule cucurbit[7]uril(CB[7]) as a model system in this study. CB[7] belongs to afamily of barrel-shaped molecular hosts, cucurbit[n]urils(CB[n]), which exhibit unique structural and chemicalmolecular recognition properties that make them suitablemolecular receptors, particularly in aqueous systems.16 TheCB[7] host is able to bind a variety of “guest” molecules such ascyclic and aromatic compounds, which are of interest forapplications including environmental pollutant sensing,17

materials design,18 and biomedical analyses.19 Confined bythe cavity volume of CB[7], only one guest molecule istypically encapsulated inside CB[7] at a given time (Figure 1c).It is known from crystallographic studies that CB[n]

undergoes structural deformations when its cavity is filled bya guest molecule.20,21 Theoretical modeling with moleculessuch as 1 and 2 also predicts pronounced changes in thediameter of CB[7] when they are included inside its cavity as aguest.22,23 These structural transformations are detected asshifts in the Raman vibrational frequencies of CB[n] asreported using a solution-based gold nanoparticle aggregate

Received: November 12, 2015Accepted: January 14, 2016Published: January 14, 2016

Letter

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© 2016 American Chemical Society 704 DOI: 10.1021/acs.jpclett.5b02535J. Phys. Chem. Lett. 2016, 7, 704−710

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system.24 It is also possible to study changes in the Ramansignatures of guest molecules in bulk (solid) complexes.25

CB[7] is known to adsorb spontaneously on gold surfacesthrough interactions with the carbonyl portals, givingorientation of its barrel axis perpendicular to the gold surface.26

Here, we use sparsely distributed gold nanoparticles on a planargold surface separated by a fractional monolayer of complexedCB[7] molecules to fabricate a NPoM geometry with a fixedgap distance of ∼0.9 nm,27 defined by the barrel height of theCB[7]. Guest molecules 1−8 (see Figure 1d) with reportedbinding affinities for CB[7] ranging from 105 to 1016 M−1 wereinvestigated in this study28 (see Supporting Information,section S2). Detailed examination of the Raman modes ofCB[7] and its complexes in the gap between particle andsurface allows statistical analysis of the host−guest system.Planar gold surfaces were prepared with a single physisorbed

layer of CB[7] of fractional coverage (see Experimental

Section). Colloidal AuNPs (diameter 100 nm) were thendrop-cast on the surfaces to form NPoMs with strong fieldenhancements in the gaps. A distinction between the filled andunfilled states of the CB[7] cavities present within the NPoMplasmonic gaps can readily be observed using dark-fieldscattering microscopy. For example, the presence of MV2+

(1) within the CB[7] cavities shows clear red-shifts in theplasmonic resonances of the NPoM system (Figure 2a,b). Thedominant plasmonic mode is a result of the coupling betweenthe AuNP and the surface. These modes are observed at ∼730nm when uncomplexed CB[7] is present in the NPoM gap,whereas this mode shifts to ∼770 nm for gaps containingCB[7]·MV2+ instead. This is a result of the higher refractiveindex of the complexed CB[7].29 The two cases were modeledusing the boundary-element method (BEM) with refractiveindices of the surrounding medium of n = 1.2 for theuncomplexed and n = 1.5 for the complexed state to

Figure 1. (a) Nanoparticle on mirror assembly. The AuNP is spaced from a gold surface by CB[7] molecules. (b) The plasmonically enhanced fieldin this construct is confined to a volume of only a few cubic nanometers. (c) The macrocyclic host cucurbit[7]uril accommodates one guest moleculeto form a host−guest complex. (d) Guest molecules investigated using the NPoM geometry: 1, methyl viologen; 2, 2,7 dimethyldiazapyrenium; 3, 1-adamantylamine; 4, N-methyl-1-adamantylamine; 5, 1-adamantane carboxylic acid; 6, (dimethylaminomethyl)ferrocene; 7, 1,1 ferrocene dimethano;and 8, ferrocene. Note: The counterion for the guests in all cases is chloride.

Figure 2. (a) Dark-field scattering spectra of NPoM with MV2+·CB[7] and unfilled CB[7] as a spacer. The refractive index change caused by theguest leads to a shift of the plasmon resonance by 30 nm. Dotted (blue) lines are the corresponding field enhancements obtained by modeling forboth cases. Vertical (red) dashed line indicates the Raman excitation wavelength (λ = 785 nm). (b) Statistical distribution of the dark-field scatteringresonant wavelengths for unfilled CB[7] (gray) and MV2+·CB[7] (blue) as spacers in the NPoM geometry. (c) From top to bottom: MV2+·CB[7]SERS spectra on single NPoM, MV2+·CB[7] complexed solid-state Raman (experiment), CB[7] solid-state Raman (experiment and simulation), andMV2+ solid-state (experiment and simulation).

The Journal of Physical Chemistry Letters Letter

DOI: 10.1021/acs.jpclett.5b02535J. Phys. Chem. Lett. 2016, 7, 704−710

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qualitatively match the experimental data. The Ramanexcitation wavelength of 785 nm is thus close to the plasmonicgap resonances, allowing the vibrational spectral positions inthe resulting SERS spectra to be compared.CB[7] has many vibrational modes in the fingerprint region

between 1100 and 1500 cm−1 with two additionallypronounced peaks that stand out at lower frequencies (Figure2c). The strongest mode at ∼440 cm−1 is a “ring scissor mode”,while the other strong mode, observed near 830 cm−1, isattributed to a “ring breathing oscillation”.30 When themacrocycle has a guest encapsulated inside its cavity, theseCB[n] breathing modes undergo distortions24,25 and provide asensitive marker to distinguish between the filled and unfilledstates of CB[7]. A typical SERS spectrum obtained from asingle NPoM (Figure 2c) contains signatures from both CB[7]and MV2+. The Raman bands observed using the NPoM are inagreement with those seen in solid reference samples for boththe host and guest molecules as well as with simulations (usingHF/3-21G; see Supporting Information, section S1).Few-molecule spectroscopy is highly sensitive to the

immediate environment of the molecules being probed,which leads to inhomogeneous broadening and subtlefluctuations in the peak positions in individual spectra (±5cm−1). Therefore, many individual spectra (on the order of 300per molecule) as well as their averaged spectra are inspected.While Raman signals are often weak in individual spectra, theyemerge as clearly visible bands in a stacked representation(Supporting Information, Figure S5).When the Raman spectra of solid CB[7] and that of CB[7]

within the NPoM geometry are compared, a new shoulder at

455 cm−1 becomes evident. This weak band is consistentlyobserved across all the samples, including for the smallerhomologue CB[5], and is likely to originate as a result ofCB[n]−Au interactions (see Supporting Information, sectionS3.2). Aside from this, shifts in the peak positions for the filledand unfilled states of CB[7] show differences depending on theguest molecule encapsulated inside the CB[7] cavity (Figure3a). An extra signal at ∼445 cm−1 is also observed for 1, 2, 7,and 8. This signal is, however, notably stronger for planarmolecules 1 and 2, which cause a pronounced ellipsoidaldeformation of the CB[7] molecule. A different behavior isseen in cases with guests 3−8, where the breathing mode at 440cm−1 is shifted to a lower wavenumber at ∼435 cm−1. Thedifferent shifts to higher or lower wavenumbers arise from theanisotropic interactions of the guest molecule with CB[7],depending on whether the guest molecules interact with onlyone or both of the two carbonyl portal rims (Figure 3b). Asummary of the shifts in the peak position of CB[7] at 440cm−1 for all the studied guests, and the fractional areas of theshifted signals, are shown in Figure 3c,d. In general, the shiftedmode is weaker (less area) than the unshifted CB[7] resonance.Guest molecules that interact with both portals exhibit shifts ofthe 440 cm−1 mode to high energy, while those that interactwith only one portal shift to lower energy.A similar trend is not observed for the mode at 830 cm−1

because this vibrational mode predominantly arises only fromthe equatorial region of the CB[7] and therefore is relativelyless affected by binding around the carbonyl portals. A slightshift toward lower frequency (∼827 cm−1) is observed in allcases, while an additional broad signal can be observed at ∼870

Figure 3. (a) Averaged SERS spectra of the CB[7] Raman modes near 440 cm−1 and showing the deconvolution (deconvoluted peaks shown withblue dashed lines), with peaks attributed to filled CB[7] colored blue. The fitted spectra for CB[7] control in the absence of guest has been overlaid(red) in each case for comparison. (b) Schematic showing characteristic trends in shifts. (c) Size of shifts and (d) areas of the shifted CB[7]vibrational mode at 440 cm−1 for each guest molecule (%, relative to unfilled CB[7]).



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cm−1 including control samples (of CB[7] and CB[5]). Thesesignals are therefore again likely to arise from the electrostaticinteraction of CB[n] with the gold surface. While similarmagnitudes for such shifts have been shown in SERS usingcolloidal gold nanoparticle aggregates,24 the different effects ofdifferent molecules have not been reported before.When probing the distribution of the complexed and

uncomplexed states of CB[7] in individual NPoMs with onlya few molecules in each plasmonic nanojunction, three differentsituations can be observed (Figure 4a): (i) the probed CB[7]molecules are unfilled, resulting in unshif ted CB[7] Ramanmodes; (ii) both filled and unfilled states of CB[7] or “mixedevents” are detected simultaneously in cases where more thanone molecule may be present in the gap; and (iii) all CB[7]molecules in the junction each contain a guest, leading to onlythe shif ted Raman modes. Representative spectra showing the400 and 800 cm−1 regions for 1 are shown in Figure 4b (seeSupporting Information, Figures S8−S10 for individual spectraof other guest molecules).For all the guest CB[7] inclusion complexes, 40%−60% of

the investigated NPoMs exhibit either only filled or onlyunfilled CBs, which strongly suggests that the SERS signal is inmany instances generated by single molecules (Figure 4). Themixed events, however, may arise in three cases: (1) More thanone molecule is probed. (2) CB[7] host−guest complexes, ingeneral, are known to be dynamic because of the “in-and-out”diffusion of guests from the CB[7] cavity. These diffusionalmovements are much faster compared to the integration time(10 s) used for measurement of SERS spectra. For example, the

lifetime of MV2+·CB[7] is reported to be 5.3 ± 0.5miliseconds.31 Therefore, it is possible that in some cases,depending on the local environment of the complexedmolecule, a temporal averaging ef fect is observed due to thisdiffusion. In these cases, signals may be obtained from the sameCB[7] molecule in a filled and unfilled state. (3) Similarly, it isalso possible that the incident laser occasionally removes theguest molecules from the CB[7] cavity, also resulting in atemporal average signal being observed. In cases 2 and 3, themixed event signals could arguably also be attributed to singlemolecules.The high number of single-molecule events suggests

somehow additional field localization addresses only a fewmolecules. Possibly this arises from imperfections and adatomsraised above the plain surface in the nanogap architecture thatcan localize light to even smaller volumes than the gap plasmonand thus play a significant role in promoting selected moleculesto produce more intense SERS signals by creating strong locallyconcentrated electric fields.32

The method presented here is thus built upon the well-established bianalyte technique, which is used to demonstratesingle-molecule sensitivity.33−35 Typically, the SERS signal of amixture of two different chemical species is investigated wherethe occurrence of only one of the two species in a spectrum isan indication that it originates from only one molecule. In thisstudy, the SERS signals from only the host molecule aremonitored, with the f illed and unf illed states of CB[7] (with aguest) now treated as two distinct species. The signatures of thetwo states are compared via the energy shift of Raman bands

Figure 4. (a) (top) Schematic illustration of distribution of the underlying composition of the CB[7] and MV2+·CB[7] mixture in the NPoM probesshowing three observable events. (bottom) Individual SERS spectra of the two predominant CB[7] Raman modes near 440 and 830 cm−1 (CB[7]modes are highlighted with gray dashed lines and MV2+ modes with red dotted lines). Spectra correspond to (i) NPoM with purely unfilled CB[7](green), (ii) a mix of filled and unfilled CB[7] (orange), and (iii) only filled CB[7] (blue). (b) Statistical representation of the number of eventsobserved. For each guest molecule, the statistics is based on the investigation of between 300−400 NPoMs with sufficiently strong SERS signals toenable identification of the relevant modes. (c) Comparison of the number of times signals from filled CB[7] is observed for single-molecule eventsonly and mixed events as a function of decreasing order of binding affinities of guests for CB[7]. Overlayed trace shows log of binding strengths ofthe guest molecules for CB[7].



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upon complexation. In addition to its simplicity, one of themain advantage of this method is that it enables every active siteto be probed and the determination of single-moleculesensitivity without relying on accurate determination of thenumber of molecules in the nanogap.Based on the known 1:1 binding stoichiometry and

association constants of the guests toward CB[7]36−39 (seeTable S1), the fraction of CB[7] that should be in a complexedstate can be estimated using the Hill equation.40 For theconcentration used in these experiments, the fraction of CB[7]expected to be in a filled state ranges from ∼90% for the weakerbinding guests such as 1 and 2 to ∼99% for the strong binders(3−5). These theoretical fractions were compared with thenumber of experimental observations of Raman signals fromfilled CB[7] in approximately 350 spectra (i.e., number ofspectra containing shifted CB[7] signals). In general, gueststhat bind to CB[7] with a strong affinity showed a higherfrequency of observations from filled CB[7]. This phenomenonis also evident in the averaged spectra, where these moleculesshow a weaker band from unfilled CB[7] (Figure 3).Furthermore, a control study using 1:10 MV 2+·CB[7] (10μM MV 2+:100 μM CB[7]) in the NPoM geometry showed asignificant increase in the number spectra containing emptyCB[7] single events compared to the number of filled CB[7]events (Supporting Information, Figure S7). The number ofmixed events observed was also lower. These results indicatethat the populations of filled and unfilled CB[7] on the goldsurfaces are likely not a random distribution of the two statesbut rather are determined by the binding affinity of the guesttoward CB[7]. This is not surprising: the formation of acomplex between a host and a guest molecule is a fundamentalprocess in supramolecular chemistry and a variety ofinteractions and energies are collectively involved.40,41 There-fore, the molecules would be expected to reside inside theCB[7] cavity governed by their respective association constants.This observation is consistent with a study investigating therecognition properties of self-assembled monolayers of CB[7]with atomic force microscopy.42

From within the hundreds of spectra analyzed for each guest,only spectra showing either only f illed CB[7] or only unf illedCB[7] (i.e., single events) were selected. More than 80% ofthese single event spectra were found to originate from onlyf illed CB[7] (Figure 4c), which is close to the aforementionedexpected range of 90−99%. However, if the single-event-onlyspectra were not preselected and all the collected spectraincluding mixed events were taken into account, the fraction offilled CB[7] was found to be lower, i.e., 45−60%. (Figure 4c).In our analyses, a mixed event is assumed to originate from onefilled CB[7] molecule and one unfilled CB[7] molecule,whereas the actual number of molecules contributing to thesignals in mixed events is unknown. Therefore, the discrep-ancies in the expected and observed number of filled CB[7]events can be reasonably well accounted for. The probableremoval of guest from the CB[7] cavity by the laser light (assuggested earlier) might also lead to the fraction of filled CB[7]being lower than expected.The intensities of the SERS signals obtained from different

NPoMs vary by roughly a factor of 10, despite the well-definedorientation of both molecules and optical fields. A majorcontributing factor to these variations in SERS intensities is thetotal number of molecules present in the enhanced field regionin the gap, which depends on the precise volume of the cavityformed by the AuNP and substrate, set by the facetting of the

NP. The 100 nm diameter AuNPs used in these experimentsexhibit typical facet diameters between ∼5 and 50 nm, resultingin up to 100-fold variations of the number of incorporatedmolecules (see Supporting Information, section S6). Assuminga tightly packed monolayer of CB[7], a small AuNP facet with a5 nm diameter can accommodate approximately 40 CB[7]molecules inside the nanogap, while a large facet of 50 nm canhold up to 4000 molecules. In this case, however, using dilutesolutions of CB[7] to form sparse monolayers, it is estimatedthat there are typically between 1 and 100 CB[7] molecules ineach NPoM gap.While perfectly spherical AuNPs would lead to similar

detection volumes in each NPoM, the actual cavity size varies asa consequence of the pronounced AuNP facetting. Driven byvan der Waals interactions, faceted nanoparticles predominantlytend to align their facets parallel to the underlying substrate43

(Supporting Information, Figure S11). This forms a nanoscopicplasmonic cavity,44 which supports standing waves with nodesof high and low field enhancements and of sizes defined by thewidth of the facet. The specific location of the molecules withinthe gap as well as the in- and out-coupling efficiency of lighttherefore becomes an important factor in determining thescattering intensity.Furthermore, the field enhancement and field distribution

within the nanogap is highly sensitive to the exact morphologyof the NPoM construct.45 Inherent nanometer-scale defectsexist on the surface of the AuNP as well as on the underlyinggold mirror. This creates extremely strong and highly localizedfields in particular areas within the gap through an additionallightning-rod effect.46 Molecules in the vicinity of these “subhotspots” potentially produce a much higher SERS intensity.Additional intensity variations are also partly caused byalignment mismatches between the laser and the symmetryaxis of the NPoM, resulting in fluctuations of the fieldenhancement.In conclusion, we show that SERS of individual NPoMs can

clearly distinguish between the different kinetic states (filled orunfilled) of the CB[7] molecules within the NPoM plasmonicnanogap. While it is common to use resonant dye moleculeswith strong Raman responses to demonstrate single-moleculeresonant SERS (SERRS),47 the results presented here confirmthat few-to-single-molecule sensitivity can also be achieved withelectronically nonresonant molecules.48 However, single-mole-cule resolution is not comprehensively achieved in all NPoMsand it is anticipated that the actual number of traced moleculesdepends on the exact morphology of the AuNP and the Ausurface.We observe subtle differences in SERS peak positions and

their respective broadening or fluctuations depending on theguest bound into the CB[7]. It is also clear that anysubstituents covalently interacting with only one of thecarbonyl portals of CB[7] exerts a considerable influence onits structure. We evidence clear effects from the symmetry ofthe guest molecules, which should now guide the developmentof more accurate theoretical models of the host−guest bindingconfiguration. Furthermore, the applicability of this system indirect statistical estimation of the number of filled CB[7] on asurface is demonstrated and may be extended to interrogatehost−guest systems in a similar manner. This concept opens upopportunities to establish host−guest chemistry as a single-molecule sensing platform useful for gas sensing, drugdetection,49 or quantitative sensing of various hazardoussubstances. Furthermore, it could be used to investigate guest



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binding properties for a variety of biologically significantmolecules such as peptides, DNA, and lipid membranes andallows real-time observation of chemical reaction kinetics.

■ EXPERIMENTAL SECTIONFlat gold surfaces were prepared using electron-beamevaporation. A 5 nm thick chromium adhesion layer wasdeposited on a silicon wafer, followed by a 70 nm thick goldfilm. Host−guest complex solutions were prepared by mixingguest molecules and CB[7] (100 μM, 1:1 stoichiometry)followed by sonication for approximately 5 min. Less water-soluble guest molecules (such as ferrocene) required longersonication times (∼30 min). Gold surfaces were functionalizedby immersion in the host−guest solution for at least 24 h toallow formation of a distorted and sparsely distributed CB[7]monolayer on the surface. Samples were then rinsed with waterand blow-dried. The 100 nm citrate-capped colloidal AuNPs(BBI solutions) were drop cast on the sample surface wherephysisorption takes place. After 5 min, excess NPs were washedaway.Raman microscopy was performed on a Renishaw inVia

Raman microscope using a 100× objective with numericalaperture NA = 0.85. To match the plasmon resonance, anexcitation wavelength of 785 nm was chosen with optical powerin the objective set to 13 mW. Integration time was 10 s. TheSERS spectra were collected on single mirror-coupled AuNPs.This was done by identifying individual AuNPs using dedicatedbright- and dark-field imaging in order to move them to thecenter of the objective to which the Raman laser was aligned aswell.SERS signals could not be observed when the Raman laser

was not centered on a nanoparticle, i.e., nothing butbackground noise was observed. Furthermore, there werecases when SERS signals were not obtained even with “on-particle” measurements. This is expected to happen if thenanoparticle being studied is not located on adsorbedmolecules as they are only sparsely distributed on the surface.Samples for reference Raman spectra of MV2+·CB[7] wasobtained by freeze-drying a 1 mM (1:1) solution of thecomplex.Optical scattering spectra were taken on an Olympus BX51

research microscope in dark-field configuration using a 100×objective with numerical aperture NA = 0.85. The samples wereilluminated using a halogen white light source, and scatteredlight was collected with a multimode fiber in confocalconfiguration attached to a cooled spectrometer.The near-field enhancement inside the nanogap was modeled

with the boundary element method using BEMAX.50,51 TheCB[7] and MV2+·CB[7] molecular spacers were modeled as alayer of constant refractive index with n = 1.2 and n = 1.5,respectively,29 between a flat Au surface and a AuNP. Thedielectric functions of Au were taken from Johnson andChristy.52 The illumination source was a broadband plane-wave, incident at an angle of 58° to account for NA = 0.85 inthe experimental setup.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jpclett.5b02535.

ITC characterization of CB[7] binding with guestmolecules 4 and 7, control studies, additional represen-

tations of the SERS spectra, SEM characterization ofsample, and estimation of number of molecules in NPoMgaps (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: [email protected].*E-mail: [email protected] Contributions¶D.O.S. and S.K.: These authors contributed equally to thiswork.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors acknowledge funding from Walters-Kundert Trust,EPSRC (EP/K028510/1, EP/G060649/1, EP/H007024/1,ERC LINASS 320503), an ERC starting investigator grant(ASPiRe 240629), EU CUBiHOLE grant, and the DefenceScience and Technology Laboratory (DSTL). S.K. thanksKrebs Memorial Scholarship (The Biochemical Society) andCambridge Commonwealth Trust for funding. F.B. acknowl-edges support from the Winton Programme for the Physics ofSustainability.

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observing single molecules complexing with cucurbit[7]uril … · abstract: in recent years,...

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